growth of chlorella vulgaris on sugarcane vinasse: the effect of anaerobic digestion pretreatment

Growth of Chlorella vulgaris on Sugarcane Vinasse:The Effect of Anaerobic Digestion Pretreatment

Sheyla Santa Isabel Marques & Iracema Andrade Nascimento &

Paulo Fernando de Almeida & Fábio Alexandre Chinalia

Received: 12 June 2013 /Accepted: 26 August 2013 /Published online: 7 September 2013# Springer Science+Business Media New York 2013

Abstract Microalgae farming has been identified as the most eco-sustainable solution forproducing biodiesel. However, the operation of full-scale plants is still limited by costs and theutilization of industrial and/or domestic wastes can significantly improve economic profits.Several waste effluents are valuable sources of nutrients for the cultivation of microalgae.Ethanol production from sugarcane, for instance, generates significant amounts of organicallyrich effluent, the vinasse. After anaerobic digestion treatment, nutrient remaining in such aneffluent can be used to grow microalgae. This research aimed to testing the potential of theanaerobic treated vinasse as an alternative source of nutrients for culturing microalgae with thegoal of supplying the biodiesel industrial chain with algal biomass and oil. The anaerobicprocess treating vinasse reached a steady state at about 17 batch cycles of 24 h producing about0.116 m3CH4 kgCODvinasse

−1. The highest productivity of Chlorella vulgaris biomass(70 mg l−1 day−1) was observed when using medium prepared with the anaerobic digestereffluent. Lipid productivity varied from 0.5 to 17 mg l−1 day−1. Thus, the results show that it ispossible to integrate the culturing of microalgae with the sugarcane industry by means ofanaerobic digestion of the vinasse. There is also the advantageous possibility of using by-products of the anaerobic digestion such as methane and CO2 for sustaining the system withenergy and carbon source, respectively.

Keywords Vinasse . Anaerobic digestion . Microalgae and biofuels

Introduction

The International Energy Agency has forecasted that the global consumption of oil will rise30 % by 2035, and that the availability of petrol will reach limiting levels a decade later [1].

Appl Biochem Biotechnol (2013) 171:1933–1943DOI 10.1007/s12010-013-0481-y

S. S. I. Marques : I. A. NascimentoLaboratório de Biologia Marinha Instituto de Biologia (IB), Universidade Federal da Bahia/Brasil,Salvador, Brazil

P. F. de Almeida : F. A. Chinalia (*)Laboratório de Biotecnologia e Ecologia de Microorganismos, Instituto de Ciências da Saúde (ICS),Universidade Federal da Bahia/Brasil, Salvador, Brazile-mail: [email protected]

Transportation was identified as the main consuming sector with about 60 % of the overallconsumption. For these reasons, several executive strategies worldwide are favoring theintroduction and commercialization of biofuels in the transportation sector [2]. Biodiesel canbe readily used by the transportation sector without significant changes in the infrastructure,and because of that, it has been estimated that the next decades will show an 80 % increaseon such biofuel production [3]. Currently, land crops are the main source of oil for biodieselproduction. Soya beans, sunflower, and rapeseed are the most common. Biodiesel annualproduction has been estimated at about 166 billion liters with a potential market value ofapproximately US$139 billion by 2016 [4]. This forecast is due to the fact that severalcountries are today commercializing blended diesel with biodiesel at ratios ranging from 5 to20 % mostly [5, 6].

There is also an environmental benefit from the use of biodiesel/diesel blends. It has beenobserved that the consumption of 20 % biodiesel/diesel blend can reduce CO2 emissions by16 % [6]. On the other hand, the use of land crops and arable soil for the production of such abiofuel may in turn offset such benefit [7]. Therefore, a different strategy must be put inplace for increasing biodiesel production in order to achieve a more sustainable developmentfor this sector. Wijffels and Barbosa [8] identified microalgae lipids as the main sustainablesource for such a goal. Approximately, 46 ton of oil ha−1 year−1 can be produced frommicroalgae, which is ten times greater than the amount of oil produced by palm tree, which isthe most productive among the conventional land crops [5]. As such, a production would notdepend on agricultural land; it would positively mitigate carbon emissions by reducing theuse of fossil fuels and fixate atmospheric carbon. Nevertheless, the production of algal oil isstill vulnerable to the influence of limiting factors such as the costs with nutrients supply andsystem operation [9]. At the moment, it is estimated that such costs may represent 50 % ofthe final algal oil prices [10, 11]. In order to improve such a scenario, it is therefore necessaryto find alternative sources of nutrients for growing such algae. A practical suggestion is toassociate algal culturing with the treatment of organic wastewater [8, 9, 12, 13]. Theintegration of algae biomass production with wastewater treatment is today recognized asthe most likely solution for economically supporting this industry [14].

Among the distinct systems used for wastewater treatment, the effluent of anaerobicdigesters has been already identified as a good medium for algal culturing [15]. This effluentis often rich in ammonia and inorganic phosphate, and it is relatively low in organic mattercontent. Additionally, this process is also known for producing significant amounts ofmethane, another renewable energy source which can be directly recycled for the operationof the system. The anaerobic process is also associated to several operational advantagessuch as comparatively low production of disposable sludge, and it consumes less energy forits operation [16–18]. The anaerobic digestion has also been suggested as the best approachfor treating vinasse, which is an organic effluent generated during the production of ethanol[19–21]. For each liter of ethanol, it is also produced about 13 L of vinasse. In Brazil alone,it was estimated a production of approximately 300 billons liters of vinasse between theyears 2011 and 2012 [22]. This effluent is currently being used for irrigating agriculturalsoil, but such practice has also been associated with depletion of fertile soil characteristics[23, 24]. Therefore, associating the production of algal biodiesel with the anaerobic treat-ment of vinasse seems to be an ideal approach for integrating the production of threerenewable biofuels: biodiesel, methane, and ethanol. Therefore, this proposal has clearcommercial advantages for it explores the potential for reducing algal biodiesel productioncosts allied with significant environmental improvements. Therefore, the aim of this researchis to evaluate the feasibility of using the effluent of an anaerobic digester treating vinasse for

1934 Appl Biochem Biotechnol (2013) 171:1933–1943

growing oil-producing algae. The overall goal is to suggest the integration between distinctprocesses resulting in the integration of biodiesel, methane, and ethanol production.

Material and Method

Experimental Design

Three distinct conditions, and their respective controls, were used in order to evaluate thefeasibility of using the effluent of an anaerobic digester treating ethanol–sugarcane vinasse forgrowing Chlorella vulgaris. Figure 1 shows that ethanol–sugarcane vinasse was diluted withtreated portions of domestic wastewater treatment plant (WWTP) effluent for standardizingvinasse chemical oxygen demand (COD) at a final value of 2 g l−1. Chemical characterizationsof the distinct waste streams used in this research are shown on Table 1. It is important tohighlight that the COD contribution of theWWTP effluent to the final mixture was insignificantcompared to the vinasse (Table 1).

After dilution, a fraction was diverted to the anaerobic digesters and another portion wasstored at −20 °C to be used as the controls for theC. vulgaris growth experiments. The anaerobicdigesters were operated until they reached a steady state performance regarding COD removaland methane production. It was only during this period that effluent samples were collected,stored (−20 °C), and later mixed for the growth experiments with C. vulgaris (Fig. 1).

Anaerobic Digestion

Ethanol–sugarcane vinasse was obtained at Agrovale S/A (Agroindústrias do Vale do SãoFrancisco S.A., Bahia, Brazil). The treated domestic WWTP effluent and anaerobic sludgewere collected at EMBASA S/A (Salvador City, Bahia, Brazil). The anaerobic sludge wasobtained from a UASB digester treating domestic effluents of Salvador City. Table 1 showssome chemical variables characterizing the effluents and also the anaerobic sludge used asinoculum. It should be highlighted that the treated domestic effluent showed a COD value325-fold smaller than what was observed with the vinasse. Treatments were carried out intriplicates using the Automatic Methane Potential Test System device from Bioprocess

Vinasse (2 g COD l-1)COD adjusted with

treated WWTP effluent

Anaerobic DigestionTreatment

Untreated vinassemixture

Growth of C vulgaris on:100% AD effluent (TV-100)50% AD effluent (TV-50)25% AD effluent (TV-25)

Growth of C vulgaris on:100% neat vinasse mixture

(NVM-100)50% neat vinasse mixture

(NVM-50)25% neat vinasse mixture

(NVM-25)

Control

Fig. 1 Diagram showing the experimental setup

Appl Biochem Biotechnol (2013) 171:1933–1943 1935

Control®, Netherland, which were operated in batch cycles of 24 h. The reactors wereinoculated with 30 % anaerobic sludge and vinasse–WWTP effluent mixture was added to afinal volume of 400 ml. After inoculation, reactors were operated with a vinasse volumetricCOD loading ratio of 1 kg m−3 day−1. Temperature was kept at 35 °C, and mixing wasprovided within an on/off cycle of 0.5 and 1.0 min, respectively. Biogas was collected andfiltered through an alkaline solution (NaOH 3 M) for capturing CO2 and H2S. Real-timemethane production was measured volumetrically. Anaerobic digestion performance wasassessed by comparing methane production kinetics and COD consumption of treatmentsand controls (Fig. 1).

Growth Kinetics of C. vulgaris

Anaerobic digester effluent was used as growth media for culturing C. vulgaris. Growthmedia were identified as TV-100, TV-50, and TV-25 % for anaerobic effluent used neat ordiluted with 50 and 75 % with WWTP effluent, respectively. The control experiment wascarried out using neat vinasse–WWTP mix (NVM-100, 50, and 25 %) without passingthrough the anaerobic digester (AD). The former growth media were thus also diluted with50 and 75 % with WWTP effluent, respectively. Table 2 shows nutrients and COD valuespresent in the influent and effluent of the anaerobic digester. Turbidity was assessed usingLaMotte® (Model 2020), and the remaining variables using the Standard Methods for theExamination of Water and Wastewater (2005) [25].

Growth trials were carried out in triplicate using Erlenmeyer flasks containing 600 ml ofstandardized medium and a 10 % volume of algal inoculums in the exponential growthphase. The flasks were kept under constant temperature and agitation (25±2 °C and 90 rpm,respectively); the aeration rate was maintained at 0.50 vvm (volume gas per volume brothper minute) of atmospheric air enriched with 2 % of CO2. Cells were incubated at a neutral

Table 1 Chemical characterization of the waste streams and the anaerobic digester influent and effluent usedin this research

Distinct waste streams used in this research Operation of AD digester

(g l−1) Sludge used asinoculum

Treated sewageeffluent

Vinasse Influent Effluent

COD 14.583 (±0.729) 0.071 (±0.004) 23.179 (±1.156) 2.06 (±0.040) 0.300 (±0.006)

NO3 0.028 (±0.002) 0.015 (±0.001) 0.026 (±0.002) 0.009 (±0.000) 0.002 (±0.000)

NO2 BDL BDL BDL BDL BDL

NH4 0.192 (±0.013) 0.005 (±0.000) 0.010 (±0.001) 0.003 (±0.000) 0.020 (±0.004)

PO4 0.022 (±0.001) 0.010 (±0.000) 0.045 (±0.002) 0.016 (±0.001) 0.014 (±0.001)

K 0.006 (±0.000) BDL 0.109 (±0.002) 0.034 (±0.005) 0.038 (±0.007)

TS 48.99 (±1.52) 0.466 (±0.03) 14.362 (±0.60) –b –b

FS 28.47 (±0.91) 0.301 (±0.01) 2.968 (±0.72) –b –b

VS 20.52 (±0.60) 0.165 (±0.02) 11.394 (±1.31) –b –b

pHa 6.7 (±0.1) 6.5 (±0.3) 3.5 (±0.1) 6.8 (±0.1) 6.7 (±0.1)

Turbidity (NTU) –b –b –b 650 (±5.2) 100 (±6.8)

BDL below detection limita Index of H+ concentrationb Not assessed


pH range (6.8±0.8), and light (140 μE m−2 s−1) was provided within a photoperiod of12:12 h light and dark cycles. Growth was monitored every 48 h by hemocytometer cellcounting and by optical density (OD). OD was determined at 680 nm using a UNICAM®Spectrophotometer model Helios Epsilon. Counting of cells per milliliter, and/or OD680

measurements were plotted against time and used for estimating growth kinetic parameters.Growth kinetic parameters were obtained in triplicates for the distinct strains, and data werecompared using ANOVA. Growth curves were adjusted using Boltzman sigmoid modeldescribed in Origin software version 7 (Origin Lab Data Analysis and Graphing Software®),which was also tested for the model validity (p≥0.05). The kinetic parameters were assessedaccording to Nascimento et al. [26]:

(a) Specific growth rate (micrometer), based on the equation μ=ln (Ny/Nx)/ (ty−tx), where Ny

andNx are the numbers of cells (N) at the start (tx) and the end (ty) of the logarithmic growthphase.

(b) Biomass productivity (Pdwt), as the dry biomass produced (g l−1 day−1) during theexponential growth phase. For Pdwt determination, samples were collected at the end ofthe exponential phase and cells were harvested by centrifugation for 5 min at 5.000 g at4 °C (Sorvall ultracentrifuge ®, Evolution RC). Supernatant discarded pellets were washedwith distilled water, freeze-dried, and the dry weight was determined gravimetrically [26].

(c) Total lipids content (Lc), reported as percentage of the total biomass (dwt%), determined byusing the chloroform/methanol approach as previously reported by Nascimento et al. [26].

(d) Volumetric Lipid productivity (Lp) calculated according to the equation Lp=Pdwt×Lc andexpressed as mg l−1 day−1 [27]. The results were compared using ANOVA and multiplerange test based on GraphPad Software Inc®.

Results and Discussion

Preliminary tests showed that vinasse was toxic to C. vulgaris at a COD concentration above4%, and according to the adopted operating design, the anaerobic digestion start-up was unstableat vinasse concentrations above 5 kg CODm3. Therefore, in order to favor a fast start-up and low

Table 2 Kinetic parameters comparing C. vulgaris grown parameters on different concentrations of anaer-obically treated vinasse (TV-100, 50 and 25 %), neat vinasse mixtures (NVM-100, 50 and 25 %), and nutrientsufficient media [26]. The table also shows chemical characterization of the biomass and lipid productivity

TV-100 TV-50 TV-25 NVM-100 NVM-50 NVM-25 Nutrientsufficientmediaa

Biomass productivity (mg l−1 day−1) 70 62 47 0 0 3 730

μ (d−1) 0.76 0.56 0.45 0 0 0.1 0.53

Lipid content (%) 23.68 24.95 26.45 20 20 20.53 28.43

Total lipid (mg l−1) 75 64 54 0 0 11 -

Lipid productivity (mg l−1 day−1) 17 15 12 0 0 0.5 204

Protein content (%) 63 65 64 0 0 63 –b

Carbohydrate content (%) 14 10 10 0 0 16 –b

a Nascimento et al. [26]b Data not available


potassium concentration, the experimental set-up was planned to test vinasse at a starting CODconcentration of 2 kg COD m3.

The anaerobic process treating vinasse reached a steady state at about 17 batch cycles of 24 hwith a COD removal rate above 80 % (Fig. 2). Methane productivity was of about 0.116 m3CH4

kgCODvinasse−1. Souza et al. [28] reported a methane productivity of 0.22 m3CH4 kgCODvinasse

−1

when their anaerobic digester was operated at 55–57 °C and with a vinasse volumetric organicload of 25–30 kg CODm3 day−1. Considering that the COD removal efficiencies were lower than70 %, the methane productivity reported by the former authors is unique report. Turkdogan-Aydino and Yetilmezsoy [29], on the other hand, reported that methane productivity of vinassevaried from 0.05 to 0.11 m3CH4 kgCODvinasse

−1 when the digester was operated with an organicvolumetric loading rate varying from 1.9 to 16 kg CODvinasse m

3 day−1. Nevertheless, it has beenobserved that anaerobic digesters operated with vinasse at a high organic volumetric loading areoften unstable during long-term operation because of accumulation of acids. Mohana et al. [19]and Goodwin et al. [30] thus suggested that for ensuring best performance, anaerobic digesterstreating vinasse should commonly be operated during the start-up period with an organicvolumetric load between 4.8 and 5.4 kg COD m3 day−1. According to the former authors,

Batch cycles

Batch cycles

CO

D r

emov

al (

%)

mlC

H4 h

-1

mlC

H4 C

OD

-1

a

b

Fig. 2 Monitoring parameters of anaerobic digesters treating vinasse: a COD removal efficiencies comparedto volumetric methane production rates and b specific methane yields of each batch cycle (24 h)


increases in organic volumetric loads would therefore be a gradual process and depen-dent on the production and accumulation of acids within the digester. As stated before,the suggested organic loading interval did not produce a stable start-up phase in thisresearch. Constant decreases in pH were observed even after correction, which stronglysuggested accumulation of acids (data not shown). The anaerobic process is directlyaffected by the type of inoculum, reactor design, and operation. The right balancebetween the hydrolytic and acetotrophic phases must be reached for ensuring thehighest performance and methane yields during the anaerobic process. Considering theconditions and inoculum quality used in this research, the best approach was to use anorganic volumetric load of 1 kg m3 day−1 of vinasse. However, it should be highlightedthat quality of the effluent should be always compared by means of performanceindicators regardless of volumetric organic loading. Thus, independent of high or loworganics, the quality of the digester effluents is analogous if the performance indicatorsare similar. For instance, Manresa et al. [31] reported that fluidized bed reactor canremove 75 % of vinasse COD (79 g l−1) with a hydraulic retention time of 8 h. On theother hand, Cabello et al. [23] observed a COD removal rate below 60 % with asimilar digester, but operated with HRT of 24 h and an organic load of 19.5 kg CODm−3 day−1. The former authors reported a methane production rate of about 1.25 mlCH4 h−1, which is lower than the observed in this work (6.5 ml CH4 h−1). Thus, thedepuration quality of their effluent is potentially lower when compared to a processwith COD removal rate above 80 % and methane yields of 0.116 m3 CH4

kgCODvinasse−1.

Maximum methane yields during high organic loading of vinasse may be achievableafter long periods of gradual adaptation. However, it is very common to observe longerhydraulic retention time as a result of the increase on vinasse organic loading [32, 33].In order to address these issues, several authors suggest a pretreatment of the vinassebefore anaerobic digestion. Sile et al. [34] tested the process of vinasse ozonation inorder to reduce the concentration of phenolic and other toxic substances. Rabelo et al.[35] reported 41 % increase in methane productivity after vinasse pretreatment withperoxidases. Therefore, in order to avoid such complications in this research, theanaerobic digesters were operated at an organic loading of 1 kg COD m3. The maingoal was to ensure success of the anaerobic process in order to test its effect on the useof treated and non-treated vinasse for growing C. vulgaris. Despite the fact that thevinasse organic loading may be considered as low, the process has achieved significantperformance as assessed by COD removal and methane yields (Fig. 2). These variablesreached steady state equilibrium at about 17 of the 25 days of operation. This is a clearindication that anaerobic treatment of vinasse was of good quality, and adaptations tothe process should make treatment of higher organic loads feasible. Adjustment in theabundance and/or activity of methanogenics may be required for avoiding accumulationof acids.

Ryan et al. [36] reported that vinasse treatment is very expensive for the ethanolindustry. Therefore, raw vinasse is commonly used for soil irrigation, but transportationcosts and soil management can often be very expensive. In addition, large discharges ofvinasse into the soil can also cause a significant environmental impact and fertility loss[36]. Therefore, anaerobic digestion of vinasse prior to irrigation has also been consid-ered as an attractive alternative for reducing organics and toxic compounds [14].Table 1 not only shows a substantial reduction on organic content but also on turbidity(84 %). However, further work is necessary in order to improve the process for copingwith 100 % vinasse in the feed. Apart from the gradual adaptation of the microbial


community, specific digester’s design may yet be required. Nevertheless, the resultsshow that anaerobic digestion is viable and productive biological process.

Growth of C. vulgaris on Vinasse

Growth experiments with C. vulgaris were prepared with distinct concentrations of anaer-obically treated and raw vinasse mixtures as the sole nutrient sources (Fig. 1). Growthkinetic parameters are shown in Table 2, and the highest value was observed with TV-100(0.76 day−1). Experiments with raw vinasse mixtures showed a negative result for NVM-100and NVM-50 %, respectively. A modest growth, however, was observed on NVM-25(0.1 day−1), which corresponds to a final vinasse concentration of 2 %. Valderrama et al.[37] reported a C. vulgaris growth of 0.46 day−1 on 10 % vinasse medium. However,Valderrama et al. [37] used a mixture of two industrial effluents with unique characteristics.They obtained vinasse from the process of producing citric/acetic acids apart from ethanol.C. vulgaris can expressively increase biomass production using acetates mixotrophically[38], and this fact may explain the contrasting algal growth rates at 2 and 10 % vinasseconcentrations. In this work, the vinasse was obtained from an ethanol producing industryand it showed to be acutely toxic to C. vulgaris at concentrations higher than 4 %. Chemicalcharacteristics are shown in Table 1.

Several authors identified that the presence of phenolic substances andmelanoidins are oftenthe cause of algal poor growth performances in the presence of untreated vinasse [19, 20, 39].Some photosynthetic microorganism (Oscillatoria boryana) can tolerate higher concentrationsby producing peroxide [40], but growth performance is still negatively affected. The addition of1 %vinasse in the growth media showed to increaseChlamydomonas reinhardii growth rates inabout 3.6-fold (from 0.25 to 0.9 day−1), but the mixture was very toxic at ratios above 5 % [41].A similar effect was observed with Spirulina maximawhich increased growth rates (from 0.1 to1.7 day−1) at vinasse concentration of 2 gCOD l−1, but it decreased sharply at higher amend-ments. In order to address such a toxicity issue, an attempt was made in culturing C. vulgaris inconjunction with Lemma minuscula, but algal biomass production was not efficient abovevinasse concentrations of 10 % [37].

In this research, anaerobic digestion contributed significantly to reducing vinasse toxicity.Algal specific growth rate on TV-100 was 1.4-fold higher (0.76 day−1) than the maximumobserved with C. vulgaris grown on nutrient sufficient medium (0.53 day−1).On the otherhand, biomass and oil productivity was of about tenfold lower compared to nutrientsufficient medium, respectively (Table 2). The growth conditions between treatments andnutrient sufficient medium reported by Nascimento et al. [26] were very similar. Theobserved difference may be the result of two factors: (a) initial biomass concentration and(b) accumulation of lipid within the cell. Cabanelas et al. [42] showed that distinct initialbiomass can generate significant differences in C. vulgaris biomass productivity with similarspecific growth rates. The distinct accumulation of reserve material in nutrient sufficientconditions is also demonstrated elsewhere [43]. The later, however, can explain only aportion of such a difference, being the former most likely the factor responsible for theobserved result. In the trial with nutrient sufficient medium, Nascimento et al. [26] startedthe experiment with an inoculum of a C. vulgaris pre-grown on the same medium. In thisresearch, inoculum of C. vulgaris was pre-grown on nutrient sufficient medium and latertransferred into the vinasse mixture. It was observed a decrease on OD (10 %) in the firsthours, and therefore, it is reasonable to accept that a portion of this inoculum was adverselyaffected by such a change. In addition, several cells may have lost their capability ofadapting and consequently growing in such a new condition. However, after adapting to


this initial shock, the remaining cells encountered the nutritional condition (nutrients percell) for growing with high specific growth rates (0.76 day−1), in spite of low biomassproductivity (Table 2). This result indicates that growth on treated vinasse mixtures must beimproved by nutrient addition and inoculum adaptation. Increases in nutrient concentrationsmay be achieved by anaerobically treating higher concentrations of vinasse, but furtherinvestigations must be carried out in regard to the effect of potassium concentration of algalproductivity. Distinct potassium concentrations can directly affect algal cells viability andgrowth rates [44]. Potassium is used for several functions related to the photosynthesismachinery, but high concentrations are known to be toxic, and they impose some metabolicadaptations [45]. In this research, potassium concentration (Table 3) was comparable to theobserved toxic levels for Microcystis spp. [44].

The highest concentration and removal of NH4 and PO4 were observed in the treatmentTV-100. Such initial concentrations were about three times higher than what was reported byValderrama et al. [37] in comparable trials using vinasse. Working with treated domesticeffluent, Cabanelas et al. [42] reported a significant growth rate (0.38 day−1) and nutrientremoval rates by C. vulgaris at NH4 and PO4 starting concentrations of 0.12 and 0.03 g l−1,respectively. The former authors reported that at the cited initial concentrations, NH4 andPO4 removal rates were of 0.009 and 0.001 g l−1 day−1, respectively. In the experiments withvinasse, the concentrations of the former nutrients were a few times lower than what wasreported with domestic effluent (Table 3), but biological removal was efficient. Table 3 also

Table 3 Chemical characterization of the media before and after the growth experiments with C. vulgaris,respectively

(gL−1) TV-100 TV-50 TV-25 NVM-25

COD

Before 0.200 (±0.014) 0.100 (±0.007) 0.050 (±0.004) 0.325 (±0.023)

After 0.119 (±0.008) 0.070 (±0.005) 0.027 (±0.002) 0.083 (±0.006)

PO4

Before 0.014 (±0.001) 0.007 (±0.001) 0.004 (±0.001) 0.004 (±0.0002)

After 0.0 0.0 0.0 0.0

K

Before 0.038 (±0.002) 0.019 (±0.001) 0.010 (±0.001) 0.008 (±0.001)

After 0.030 (±0.002) 0.015 (±0.001) 0.009 (±0.001) 0.012 (±0.001)

NO3

Before 0.002 (±0.0) 0.001 (±0.0) 0.000 0.002 (±0.0)

After 0.0 0.0 0.0 0.0

NH4

Before 0.020 (±0.0) 0.010 (±0.0) 0.005 (±0.0) 0.000 (±0.0)

After 0.0 0.0 0.0 0.0

pH

Before 6.7 (±0.1) 6.7 (±0.1) 6.7 (±0.1) 6.7 (±0.1)

After 7.0 (±0.1) 7.5 (±0.1) 7.3 (±0.1) 7.0 (±0.1)

Turbidity (NTU)

Before 85 (±1.31) 43 (±0.95) 21 (±0.38) 65 (±1.14)

After 0.8 (±0.06) 0.4 (±0.03) 0.2 (±0.02) 0.8 (±0.06)

TV treated vinasse; NVM neat vinasse mixture (non-treated)


shows that C. vulgaris have considerably reduced turbidity values, suggesting furthercomplementing action to the treatment obtained with the anaerobic digestion (Tables 1and 3). It was observed a very small change on COD concentrations (Table 3), suggestingvery little contribution of mixotrophic activity to the observed growth rates. As expected,potassium concentrations remain statistically unchanged during vinasse treatment with C.vulgaris. Further investigation is needed in order to address this issue. However, thecombination of anaerobic digestion and C. vulgaris cultivation showed significant reductionof vinasse organics, N and P contents in negative correlation with methane and algal oilproduction.

Conclusion

Vinasse used in this research was highly toxic to C. vulgaris at concentrations above 4 %.Among the treatments, anaerobically treated vinasse (at about 8.6 % of the feed) generatedthe highest values of specific growth rates (0.76 day−1, respectively). These values aresignificantly higher than what was observed with nutrient sufficient medium (0.53 day−1)incubated at the same condition. Algal biomass productivity was lower between the vinassetrials and nutrient sufficient media, and such result was associated to lower inoculumviability and cellular accumulation of lipids. A modest growth (0.1 day−1) was also observedwith untreated vinasse at about 1.4 %. C. vulgaris have significantly reduced N, P, andturbidity values from the media, corroborating the idea of using them as a tertiary process oftreatment for effluents. The anaerobic process treating vinasse reached a steady state at about17 batch cycles of 24 h with a COD removal rate above 80 %. Methane productivity was ofabout 0.116 m3CH4 kgCODvinasse

−1. The anaerobic process was not only efficient inremoving COD but it also reduced vinasse turbidity in 84 %.

Acknowledgments The authors would like to acknowledge the financial support from the Brazilian Re-search Councils: grant 551134/2010-0 from CNPq and grant PET004/2103 from CAPES/FAPESB and alsothe donation of vinasse by Agrovale S/A, Brazil.

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